Fetal RHD genotyping from maternal plasma may be used with confidence, although additional polymorphisms for confirmation of fetal DNA should be included for 100 percent predictive value (instead of 99.6%).

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a relatively rare condition (1/1000–1/2000) that was granted orphan status by the European Medicines Agency in 2011. Clinical consequences of FNAIT, however, may be severe. A thrombocytopenic fetus or new-born is at risk of intracranial hemorrhage that may result in lifelong disability or death. Preventing such bleeding is thus vital and requires a solution. Anti-HPA1a antibodies are the most frequent cause of FNAIT in Caucasians. Its pathogenesis is similar to hemolytic disease of the newborn (HDN) due to anti-RhD antibodies, but is characterized by platelet destruction and is more often observed in the first pregnancy. In 75 % of these women, alloimmunization by HPA-1a antigens, however, occurs at delivery, which enables development of antibody-mediated immune suppression to prevent maternal immunization. As for HDN, the recurrence rate of FNAIT is high. For advancing diagnostic efforts and treatment, it is thereby crucial to understand the pathogenesis of FNAIT, including cellular immunity involvement. This review presents the current knowledge on FNAIT. Also described is a program for HPA-1a screening in identifying HPA-1a negative pregnant women at risk of immunization. This program is now performed at the Institute of Hematology and Transfusion Medicine in cooperation with the Department of Obstetrics and Gynecology of the Medical Centre of Postgraduate Education in Warsaw as well as the UiT The Arctic University of Norway.

BACKGROUND Blood donors exhibiting a weak D or DEL phenotypical expression may be mistyped D negative by standard serology hence permitting incompatible transfusion to D negative recipients. Molecular methods may overcome these technical limits. Our aim was to estimate the frequency of RHD alleles among the apparently D negative Polish donor population and to characterize its molecular background. STUDY DESIGN AND METHODS Plasma pools collected from 31,200 consecutive Polish donors typed as D negative were tested by real-time PCR for the presence of RHD specific markers located in the intron 4, exons 7 and 10. RHD positive individuals were characterized by PCR or cDNA sequencing and serology. RESULTS Plasma cross-pool strategy revealed 63 RHD positive donors harboring RHD*01N.03(n=17), RHD*15(n=12), RHD*11(n=7), RHD*DEL8(n=3), RHD*01W.2(n=3), RHD-CE(10)(n=3), RHD*01W.3, RHD*01W.9, RHD*01N.05, RHD*01N.07, RHD*01N.23, RHD(IVS1-29G>C) and two novel alleles: RHD*(767C>G)(n=3), RHD*(1029C>A). Among 47 cases available for serology, 27 were shown to express the D antigen CONCLUSION 1/ Plasma cross-pool strategy is a reliable and cost-effective tool for RHD screening. 2/ 0.2% of D negative Polish donors carry some fragments of the RHD gene; all of them were C or E positive. 3/ Almost 60% of the detected RHD alleles may be potentially immunogenic when transfused to a D negative recipient.

BACKGROUND Fetuses whose mothers have produced antibodies to red blood cell (RBC) or platelet antigens are at risk of being affected by hemolytic disease or alloimmune thrombocytopenia, respectively, only if they inherit the incompatible antigen. Noninvasive diagnosis of the fetal antigen is employed for management of immunized pregnancies, but the specific detection of SNPs, encoding the majority of antigens, in maternal plasma is still a challenge. We applied targeted next‐generation sequencing (NGS) to predict the fetal antigen based on the detection of fetomaternal chimerism. METHODS AND MATERIALS The DNA of 13 pregnant women (with anti‐K [3] anti‐k [1], anti‐Fya [1], anti‐D + C + Jka [1], anti‐D + E + K [1], anti‐HPA‐1a [1], anti‐HPA‐3b [1], anti‐HPA‐5b [1], and nonimmunized [3]) was sequenced using primers for regions encoding RhD, RhC, Rhc, RhE/e, K/k, Fya/b, Jka/b, MN, Ss, and HPA‐1, 2, 3, 5, 15, 4 X‐polymorphisms on the Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RESULTS NGS results were in agreement with the phenotype/genotype of women and their neonates (except for the unsuccessful detection of MN and RhC). NGS determined fetal allele chimerism for K, k, Fya, Fyb, Jka, Jkb, S, RhE (from 0.42% to 6.08%); RhD, Rhc (100%); HPA‐1a, −2b, −3a, 3b, −5b, −15a, 15b (from 0.23% to 4.11%). NGS revealed fetal chimerism for incompatible antigens (from 0.7% to 4.8%) in 7 immunized cases, excluded in 3 (with anti‐K, anti‐Fya, anti‐HPA‐3b). CONCLUSION The designed NGS predicts the fetal RBC and platelet antigen status universally in cases with various clinically significant antibodies as well as providing confirmation of the presence of fetal DNA. However, some improvement of the unsuccessful primers is required.

Background and Objectives Non‐invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non‐invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi‐national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. Materials and Methods We reviewed the literature covering validation for in‐vitro diagnostic (IVD) assays in general and for non‐invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co‐authors. Results In relation to Annex VIII of the In‐Vitro‐Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non‐invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti‐D prophylaxis for non‐immunized RhD negative women should be placed under risk class C. To ensure high quality of non‐invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre‐analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. Conclusion These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in‐house assays.